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United States Patent |
5,559,009
|
Chandy
,   et al.
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September 24, 1996
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Voltage-gated potassium channel gene, KV1.7, vectors and host cells
comprising the same, and recombinant methods of making potassium
channel proteins
Abstract
This disclosure relates to the identification of a new voltage-gated
potassium channel gene, Kv1.7, which is expressed in pancreatic
.beta.-cells. The invention utilizes this new potassium channel for assays
designed to identify extrinsic materials with the ability to modulate said
channel for the development of therapeutics effective in the treatment of
non-insulin-dependent diabetes mellitus.
Inventors:
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Chandy; Kanianthara G. (Laguna Beach, CA);
Kalman; Katalin (Irvine, CA);
Chandy; Grischa (Irvine, CA);
Gutman; George A. (Costa Mesa, CA)
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Assignee:
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The Regents of The University of California (Oakland, CA)
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Appl. No.:
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288405 |
Filed:
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August 10, 1994 |
Current U.S. Class: |
435/69.1; 435/252.3; 435/254.11; 435/320.1; 435/325; 536/23.5 |
Intern'l Class: |
C12N 015/12; C12N 015/63; C12N 005/10; C12N 001/21 |
Field of Search: |
536/23.5
435/69.1,320.1,240.2,252.3,254.11
|
References Cited
Other References
Bell, G. I., et al., "Sequence of the Human Insulin Gene", Nature,
284:26-32, (1980).
Horst-Sikorska, W., et al., "Prevalence of beta allele of the insulin gene
in type II diabetes mellitus", Human Genetics, 93:325-328, (1994).
Herman, W. H., et al., "Abnormal Insulin Secretion, Not Insulin Resistance,
Is the Genetic or Primary Defect of MODY in the RW Pedigree", Diabetes,
43:40-46, (1994).
Boyd, III, A. E., "Sulfonylurea Receptors, Ion Channels, and Fruit Flies",
Diabetes, 37:847-850, (1988).
Rajan, A. S., et al., "Ion Channels and Insulin Secretion", Diabetes Care,
13(3):340-363, (1990).
Misler, S., et al., "A Metabolite-regulated potassium channel in rat
pancreatic B cells", Proc. Nat'l. Acad. Sci., 83:7119-7123, (1986).
Petersen, O. H. et al., "Electrophysiology of the Pancreas", Physiological
Reviews, 67(3):1054-1116, (1987).
Ashcroft, F. M., "Adenosine 5'-Triphosphate-Sensitive Potassium Channels",
Ann. Rev. Neurosci., 11:97-118, (1988).
Dukes, I., et al., "Dependence on NADH Produced during Glycolysis for
.beta.-Cell Glucose Signaling", The Journal of Biological Chemistry,
269(15):10979-10982, (1994).
Cook, D. L., et al., "Pancreatic B Cells are Bursting, but how?", Trends
Neurosci, 14:411-414, (1991).
Smith, P. A., et al., "Delayed Rectifying and Calcium-activated K+ Channels
and Their Significance for Action Potential Repolarization in Mouse
pancreatic .beta.-Cells", J. Gen. Physiol., 95:1041-1059, (1990).
Smith, P. A., et al., "Simultaneous recordings of glucose dependent
electrical activity and ATP-regulated K+-currents in isolated mouse
pancreatic .beta.-cells", FEBS, 261(1):187-190, (1990).
Atwater, I., et al., "Properties of the Ca-Activated K+ Channel in
Pancreatic .beta.-Cells", Cell Calcium, 4:451-461, (1983).
Ammala, C., et al., "Inositol trisphosphate-dependent periodic activation
of a Ca.sup.2+ -activated K.sup.+ conductance in glucose-stimulated
pancreatic .beta.-cells", Nature, 353:849-853, (1991).
Worley III, J. F., et al., "Endoplasmic Reticulum calcium Store Regulates
Membrane Potential in Mouse Islet .beta.-Cells", The Journal of Biological
Chemistry, 269 (20):14359-14362, (1994).
Bertolli, A., et al. "Activation and Deactivation Properties of Rat Brain
K.sup.+ Channels of the Shaker-Related Subfamily," European Biophysics
Journal, 23:379-384 (1994).
Betsholtz, C., et al., "Expression of voltage-gated K.sup.+ Channels in
Insulin-Producing Cells: Analysis by Polymerase Chain Reaction," FEBS
Letters, 263(1):121-126 (1990).
Chandy, K. G., et al., "Nomenclature for Mammalian Potassium Channel
Genes," Trends in Pharmacological Sciences, 14:434 (1993).
Philipson, L. H., et al., "Sequence and Functional Expression in Xenopus
oocytes of a Human Insulinoma and Islet Potassium Channel," Proc. Nat'l.
Acad. of Sci. USA, 88:53-57 (1991).
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Primary Examiner: Draper; Garnette D.
Assistant Examiner: Fitzgerald; David L.
Attorney, Agent or Firm: Dreger; Walter H.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part of U.S. application Ser. No. 08/207,401,
filed Mar. 4, 1994, abandoned.
Claims
What is claimed is:
1. An isolated DNA molecule encoding a Kv1.7 potassium channel protein
having the amino acid sequence shown in SEQ ID NO: 10.
2. A DNA molecule according to claim 1, having the nucleotide sequence
shown in SEQ ID NO: 9.
3. A replicable vector comprising a DNA molecule according to claim 1,
4. A vector according to claim 3, wherein said vector is an expression
vector.
5. A cultured cell comprising heterologous DNA having the sequence of the
DNA molecule of claim 1.
6. A cell according to claim 5, wherein said cell is a mammalian cell.
7. A method of producing a Kv1.7 potassium channel protein comprising the
steps of introducing a DNA molecule according to claim 1 into a suitable
expression system and effecting the expression of said molecule, whereby
said potassium channel protein is produced.
Description
Reference is hereby made to the following related applications: Ser. No.
07/955,916, filed Oct. 2, 1992, now U.S. Pat. No. 5,372,702, and Ser. No.
08/170,418, filed Dec. 20, 1993, and to their parent applications, all of
which being hereby expressly incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to the identification of a new voltage-gated
potassium channel gene, Kv1.7, which is expressed in the rat and hamster
insulinoma cell lines, RINm5F and HIT, respectively. Since voltage-gated
potassium channels modulate insulin secretion from pancreatic
.beta.-cells, selective Kv1.7 blockers would be expected to increase
insulin release and thereby reduce hyperglycemia associated with
non-insulin-dependent diabetes mellitus.
The present invention is also directed toward assays for testing extrinsic
materials for their ability to block the Kv1.7 channel, and thereby exert
an effect on insulin secretion from .beta.-cells. To this end, we have
generated an expression construct, containing the coding region of the
Kv1.7 gene and have demonstrated that this gene, when expressed in Xenopus
oocytes, encodes a voltage-dependent, rapidly-activating, non-inactivating
delayed rectifier-type channel that is both tetraethylammonium- and
4-aminopyridine-resistant. This construct can now be used for the
development of mammalian cell lines expressing this channel; such cell
lines could be used in high-throughput screening assays of extrinsic
materials.
BACKGROUND OF THE INVENTION
Mammalian cell membranes perform very important functions relating to the
structural integrity and activity of various cells and tissues. Of
particular interest in membrane physiology is the study of trans-membrane
ion channels which act to directly control a variety of physiological,
pharmacological and cellular processes. Numerous ion channels have been
identified including calcium (Ca), sodium (Na) and potassium (K) channels,
each of which have been analyzed in detail to determine their roles in
physiological processes in vertebrate and insect cells.
A great deal of attention has recently been focused on the potassium
channel because of its involvement in maintaining normal cellular
homeostasis. A number of these potassium channels open in response to
changes in the cell membrane potential. Many voltage-gated potassium
channels have been identified and are distinguishable based on their
electrophysiological and pharmacological properties. An extended family of
at least twenty genes have been isolated, each encoding functionally
distinct voltage-gated potassium channels, and each with a unique tissue
distribution pattern. Several of these have been shown to be involved in
maintaining the cell membrane potential and controlling the repolarization
of the action potential in neurons, muscle and pancreatic .beta.-cells.
Potassium currents have been shown to be more diverse than sodium or
calcium currents and also play a role in determining the way a cell
responds to an external stimulus. The diversity of potassium channels and
their important physiological role highlights their potential as targets
for developing therapeutic agents for various diseases.
Type II or non-insulin-dependent diabetes (NIDDM) is a chronic and
debilitating disorder affecting at least 5% of the human population (Bell,
G. I. et al., 1980, Nature 284:26 and Horst-Sikorska, W. et al., 1994,
Hum. Genet. 93:325). NIDDM, manifested as fasting hyperglycemia, results
either from a defect in insulin release from pancreatic .beta.-cells or
from the inability of peripheral tissues to respond appropriately to
insulin (Bell, G. I. et al., 1980, supra, Horst-Sikorska, W. et al., 1994,
supra and Herman, W. H. et al., 1994, Diabetes 43:40).
Current therapeutic management of this disease is based primarily on the
use of drugs (sulfonylurea compounds) that enhance insulin release by
selectively modulating K.sub.ATP channels (Boyd III, A. E., 1988, Diabetes
37:847, Rajan, A. S. et al., 1990, Diabetes Care 13:340, Misler, S. et
al., 1986, Proc. Natl. Acad. Sci USA 83:7119, Petersen, O. H. and Findlay,
I., 1987, Physiol. Rev. 67:1054 and Ashcroft, F. M., 1988, Ann. Rev.
Neurosci. 11:97). Hypoglycemia is a frequent side effect of such
anti-diabetic therapy because these drugs, mimicking the action of
glucose, induce membrane depolarization of .beta.-cells (Bell, G. I. et
al., 1980, supra, Horst-Sikorska, W. et al., 1994, supra and Herman, W. H.
et al., 1994, supra, Boyd III, A. E., 1988, supra, Rajan, A. S. et al.,
1990, supra, Misler, S. et al., 1986, supra, Petersen, O. H. and Findlay,
I., 1987, supra, Ashcroft, F. M., 1988, supra, Dukes, I. et al., 1994, J.
Biol. Chem. 269:10979, Cook, D. L. et al., 1991, Trends Neurosci. 14:411,
Smith, P. A. et al., 1990, J. Gen. Physiol. 95:1041, Smith, P. A. et al.,
1990, FEBS Lett. 261:187, Atwater, I. et al., 1983, Cell Calcium 4:451,
Ammala, C. et al., 1991, Nature 353:849 and Worley III, J. F. et al.,
1994, J. Biol. Chem. 269:12359). Sulfonylurea-induced insulin release,
therefore, occurs in a glucose-independent manner. A glucose-dependent
insulin secretagogue could potentially avoid the debilitating side effect
of hypoglycemia, and would therefore be extremely useful.
Another form of treatment in severe long-standing NIDDM is insulin
replacement. This approach, although effective, is time-consuming,
expensive and requires the administration of painful injections often many
times daily. To say the least, NIDDM patients would welcome a more
effective treatment with fewer side effects. An understanding of the
mechanisms responsible for insulin secretion may help identify new targets
for the development of such novel anti-diabetic drugs.
Transmembrane ion channels are the primary elements that transduce signals
in pancreatic .beta.-cells, resulting in the release of insulin (Boyd III,
A. E., 1988, supra, Rajan, A. S. et al., 1990, supra, Misler, S. et al.,
1986, supra, Petersen, O. H. and Findlay, I., 1987, supra , Ashcroft, F.
M., 1988, supra, Dukes, I. et al., 1994, supra, Cook, D. L. et al., 1991,
supra, Smith, P. A. et al., 1990, J. Gen. Physiol. 95:1041, Smith, P. A.
et al., 1990, FEBS Lett. 261:187, Atwater, I. et al., 1983, supra, Ammala,
C. et al., 1991, supra and Worley III, J. F. et al., 1994, supra.). In
response to an elevation in external glucose, the .beta.-cell membrane
slowly depolarizes (phase I). This metabolic coupling appears to be due to
an increase in cytosolic ATP, which results in the closure of
ATP-sensitive potassium (K.sub.ATP) channels. The membrane depolarization
in turn initiates sinusoidal bursts of calcium action potentials (phase
II), during which intracellular calcium rises, triggering insulin
secretion (Boyd III, A. E., 1988, supra, Rajan, A. S. et al., 1990, supra,
Misler, S. et al., 1986, supra, Petersen, O. H. and Findlay, I., 1987,
supra, Ashcroft, F. M., 1988, supra, Dukes, I. et al., 1994, supra, Cook,
D. L. et al., 1991, supra, Smith, P. A. et al., 1990, J. Gen. Physiol.
95:1041, Smith, P. A. et al., 1990, FEBS Lett. 261:187, Atwater, I. et
al., 1983, supra, Ammala, C. et al., 1991, supra and Worley III, J. F. et
al., 1994, supra). Voltage-gated potassium channels have been suggested to
play a critical role in repolarizing the membrane after each of these
calcium spikes.
Alteration in any of these ionic signalling events could interfere with
insulin release and result in hyperglycemia. Overexpression of
voltage-gated potassium channels, for example, might be expected to
excessively hyperpolarize the membrane following each calcium spike and
thereby inhibit the reopening of voltage-gated calcium channels with the
reduction in calcium entry leading to diminished insulin release and
hyperglycemia. We have therefore focused our attention on identifying the
pancreatic islet cell voltage-gated potassium channel.
SUMMARY OF THE INVENTION
The present invention relates to the identification of a new voltage-gated
potassium channel gene, Kv1.7, which is expressed in the rat and hamster
insulinoma cell lines, RINm5F and HIT, respectively. Thus, the present
invention is predicated on the identification and characterization of a
marker molecule in pancreatic .beta.-cells that modulates insulin release
and that leads to a general therapeutic target for NIDDM. This predicate,
in combination with the generation of an expression construct, makes
possible the development of an assay to identify extrinsic materials
possessing the ability to selectively modulate the marker and thereby
modulate insulin secretion.
Having established a link between potassium channel function and insulin
secretion from pancreatic .beta.-cells as a predicate of the present
invention, it follows that the present invention is further directed to
associated consequential aspects including assays for testing extrinsic
materials for their ability to modulate the Kv1.7 potassium channel, and
thereby exert an effect on insulin secretion from pancreatic .beta.-cells.
The present invention is further directed to a method for treating NIDDM in
an organism manifesting said disease comprising contacting said organism
with an extrinsic material having a modulating effect on Kv1.7 potassium
channels, such materials identified by employing the assay system
described supra.
The present invention is further directed to kits containing the associated
structure, reagents and means to conduct screening assays as described
supra.
Further, the present invention is directed to the foregoing aspects in all
their associated embodiments as will be represented as equivalents within
the skill of those in the art.
The present invention is thus directed to the management and control of
NIDDM including selectively screening for, preferably selective,
modulators of Kv1.7 potassium channels for use as a therapeutic.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A represents the mouse Kv1.7 coding sequence which is indicated by
the two stippled boxes. The six bars within these regions indicate the
putative membrane-spanning domains S1 through S6. Restriction sites are
indicated as follows: BglII (B) , EcoRI (E) , PstI (P) and SacI (S) . The
order of restriction sites was determined by single, partial and double
digests and by DNA sequencing. Also indicated is a comparison of the
genomic sequence of mouse Kv1.7 (SEQ ID NOS: 1 and 3) with that of mouse
(mKv1.7 ) (SEQ ID NO: 5) and hamster (haKv1.7 ) (SEQ ID NO: 7) cDNAs
showing the splice donor and acceptor sites which form the boundaries of
the single intervening sequence.
FIG. 1B shows the deduced amino acid sequence (SEQ ID NO:10) of mouse
Kv1.7. The six putative membrane-spanning domains (S1 through S6) and
pore-forming region (P) are also indicated. Potential sites of
post-translational modification are shown as follows: N-glycosylation (*);
tyrosine kinase (TY-K) and protein kinase C (PKC). Every tenth residue is
indicated by a dot above. The hydrophobic core of this protein shares
considerable sequence similarity with other Shaker-family channels, while
the intracellular N- and C-termini and the external loops between S1/S2
and S3/S4 show little conservation.
FIG. 2 shows Northern blot analysis of total RNA isolated from the hamster
insulinoma HIT cell line (H) and rat insulinoma RINm5F cell line (R). The
probe used was a PstI/SacI fragment from the Kv1.7-specific 3'
untranslated region of the Kv1.7 cDNA. Molecular weight markers are also
presented. In both cases a 2.0 kilobase band is observed.
FIGS. 3A, 3B and 3C present the complete nucleotide sequence (SEQ ID NO:9)
of the entire coding region for the mouse Kv1.7 gene as compared to
portions of the human Kv1.7 gene sequence(SEQ ID NOS:11-19). The mouse
Kv1.7 (SEQ ID NO:9) sequence is presented on the top line whereas the
bottom line represents the corresponding human Kv1.7 sequence (SEQ ID
NOS:11-19). Dashes (-) in the human sequence represent nucleotides that
are identical to those presented in the mouse sequence. Open spaces in the
human sequence represent regions for which no sequence data is available.
FIG. 4 shows the deduced order of two potassium channel genes, hKv1.7 and
hKv3.3, on human chromosome 19.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
By the term "extrinsic material" herein is meant any entity that is not
ordinarily present or functional with respect to the Kv1.7 potassium
channel and/or pancreatic islet cells and that affects the same. Thus, the
term has a functional definition and includes known, and particularly,
unknown entities that are identified to have a modulating effect on Kv1.7
channel expression, and/or the associated pancreatic islet cells.
By the term "modulating effect", or grammatical equivalents, herein is
meant both active and passive impact on the Kv1.7 potassium channel and/or
pancreatic islet cells. These include, but shall not be construed as
limited to, blocking or activating the channel or the function of the
channel protein to materials that ordinarily permeate therethrough,
reducing or increasing the number of ion channels per cell and use of
secondary cell(s) or channel(s) to impact on a primary abnormal cell.
B. Detailed Description
A new Shaker-related potassium channel gene. We now have identified a novel
potassium channel gene, Kv1.7, which belongs to the Shaker-subfamily of
genes. A restriction map of a 6.4 kilobase EcoRI DNA fragment containing
the entire mouse Kv1.7 coding region is shown in FIG. 1A. Unlike all other
known mammalian Shaker-related genes (Kv1.1-Kvl.6) that have intronless
coding regions (Swanson, R. A. et al., 1990, Neuron 4:929, Chandy, K. G.
et al., 1990, Science 247:973, Douglass, J. et al., 1990, J. Immunol.
144:4841, Roberds, S. L. and Tamkun, M. M., 1991, Proc. Natl. Acad. Sci.
USA 88:1798, Tamkun, M. M. et al., 1991, FASEB J. 5:331, Migeon, M. B. et
al., 1992, Epilepsy Res. 6(supp.):173 and Shelton, P. A. et al., 1993,
Receptors and Ion Channels 1:25), the protein coding region of mouse Kv1.7
is interrupted by a single 1.9 kilobase intron whose splice sites are
shown in FIG. 1A. The deduced mouse Kv1.7 protein (SEQ ID NO:10) consists
of 532 amino acids and contains six putative membrane-spanning domains,
S1-S6 (FIG. 1B). The upstream exon encodes the amino terminus and the
first transmembrane segment (S1), while the remainder of the coding
sequence is contained within the downstream exon.
Expression of Kvl. 7 in pancreatic .beta.-cells. Northern blot assays using
a Kv1.7-specific 3'-NCR probe revealed a strongly hybridizing 2 kilobase
band in the rat and hamster insulinoma lines, RINm5F and HIT (see FIG. 2).
RINm5F and HIT cells are neoplastic versions of pancreatic .beta.-cells
and can secrete insulin in response to glucose challenge like their normal
counterparts. These cells have been widely used as models for normal
pancreatic .beta.-cells. We have also demonstrated the presence of Kv1.7
mRNAs in these cells by PCR analysis, which we confirmed by sequencing (a
portion of the hamster sequence is shown in FIG. 1). Betsholtz, C. et al.,
1990, FEBS Lett. 263:121 have also used PCR to amplify a short segment of
Kv1.7 cDNA spanning the S5/S6 region from mouse (MK-6), rat (RK-6) and
hamster (HaK-6) insulin-producing cells. Our sequence is identical to
their MK-6 sequence in the short region of overlap, except for four single
nucleotide changes.
These results led us to hypothesize that Kv1.7 is expressed in normal
pancreatic islet .beta.-cells, and may play an important role in the
electrical events regulating insulin release, making it a potential
therapeutic target for NIDDM. To test this idea, we provided
Kv1.7-specific DNA probes to Dr. Julie Tseng-Crank at Glaxo, for in situ
hybridization on histological sections of pancreata from normal and
diabetic db/db mice. In confirmation of our prediction, Dr. Tseng-Crank
found that Kv1.7 mRNA was present in both normal and diabetic islet cells.
Electrophysiological and pharmacological properties of Kv1.7. To study the
properties of this channel, we generated an expression construct in which
the intron was spliced out, along with the 5'- and 3'-non-coding
sequences. This construct, when expressed in Xenopus oocytes, encodes a
channel which is voltage-dependent, rapidly-activating and
non-inactivating, and is TEA- and 4AP-resistant.
Chromosomal location of Kv1.7 in humans. DNA probes from mouse Kv1.7 and
Kv3.3 were isolated and sent to the Human Genome (Chromosome 19) Center at
Lawrence Livermore laboratory. We had previously demonstrated that Kv1.7
and Kv3.3 were located on human chromosome 19 (Ghanshani, S. et al., 1992,
Genomics 12:190 and McPherson et al., 1991, in Eleventh International
Workshop on Human Gene Mapping), and needed more specific localization.
Dr. Mohrenweiser's group used these mouse probes to isolate human Kv1.7-
and Kv3.3containing cosmid clones from a chromosome 19 library, and then
used the human cosmids as fluorescent-probes for in situ hybridization
experiments to map both genes to human 19q13.3-13.4. The idiogram of human
chromosome 19 shown in FIG. 4 indicates that Kv1.7 (KCNA7) is located
centromeric of Kv3.3 (KCNC3). Genes for both glycogen synthase (GSY) and
the histidine-rich calcium protein (HRC) also map centromeric of Kv3.3,
but the order of Kv1.7, HRC and GSY could not be resolved by fluorescence
in situ hybridization experiments. Studies by S. Elbein and colleagues,
however, have placed HRC approximately 4 cM centromeric to GSY.
NIDDM is heterogeneous in its etiology, and families have been described in
which the disease is associated with mutations in either glucokinase
(chromosome 7) or a gene closely linked to adenosine deaminase (chromosome
20) (Vaxillaire, M. et al., 1994, Diabetes 43:389, Froguel, P. et al.,
1993, N. Eng. J. Med. 328:697 and Bell, G. I. et al., 1991, Proc. Natl.
Acad. Sci. USA 88:1484). Additional forms of NIDDM exist which are not
linked to either of these genes (Vaxillaire, M. et al., 1994, supra,
Froguel, P. et al., 1993, supra and Bell, G. I. et al., 1991, supra) and
recent studies suggest that a locus predisposing to diabetes exists at
human chromosome 19q13.3. First, in a large group of unrelated patients in
Finland, a polymorphism of the GSY gene is associated with the development
and severity of NIDDM (Groop, L. C. et al., 1993, N. Eng. J. Med. 328:10
and Vestergaard, et al., 1993, J. Clin. Invest. 91:2342). However, there
was no evidence for structural defects in the GSY gene or alterations in
the total level of GSY protein in these patients, indicating that
expression of this gene was unaltered, and suggesting that GSY may only be
a marker for another gene on 19q13.3 (Groop, L. C. et al., 1993, supra and
Vestergaard, et al., 1993, supra). More recent studies using polymorphic
markers in this region exclude the GSY gene as a candidate (Vaxillaire, M.
et al., 1994, supra, Froguel, P. et al., 1993, supra, Bell, G. I. et al.,
1991, supra, Groop, L. C. et al., 1993, supra and Vestergaard, et al.,
1993, supra), and suggest that a diabetic susceptibility gene may lie
centromeric to HRC and away from GSY. The localization of the islet cell
potassium channel gene, Kv1.7 (KCNA7), to human 19q13.3 and its
overexpression in diabetic islets therefore make it a candidate; Kvl.5 was
excluded because it is on human chromosome 12p13 (Curren, M. et al., 1992,
Genomics 12:729 and Attali, B. et al., 1993, J. Biol. Chem. 268:24283),
and is not found in islet cells (see above). Thus, Kv1.7 may be a
candidate gene for some inherited forms of NIDDM associated with impaired
insulin secretion.
Sequence analysis of the human Kv1.7 gene. Numerous partial human Kv1.7
cDNA clones have been isolated using the mouse Kv1.7 cDNA as a probe and
sequence data from the human Kv1.7 gene have been obtained. Partial human
Kv1.7 sequences, (SEQ ID NOS:11-19) in comparison to the sequences of the
mouse Kv1.7 coding region, (SEQ ID NO:9) is shown in FIGS. 3A and 3B. The
sequence information in FIGS. 3A and 3B demonstrates that portions of the
human Kv1.7 gene possess a great deal of homology with that of the mouse
Kv1.7 gene.
Kv1.7-selective blockers could function as glucose-dependent insulin
secretagogues. We have shown that Kv1.7 is a novel Shaker-related gene
encoding a rapidly activating, non-inactivating, TEA-resistant
voltage-gated potassium channel expressed in pancreatic .beta.-cells.
Voltage-gated potassium channels with properties similar to Kv1.7 have
been reported to regulate membrane repolarization following each calcium
spike during phase II of insulin secretion. A Kv1.7 blocker would
therefore be expected to lead to glucose-dependent modulation of insulin
release, potentially avoiding the debilitating side effect of
hypoglycemia. Such drugs would have wide therapeutic use in the management
of NIDDM.
Use of the Kv1.7 expression construct to identify Kv1.7-specific
glucose-dependent insulin secretagogues. The Kv1.7 expression construct
described above has been successfully used to generate functional
potassium channels with unique properties. This construct or related ones
can be used for expression of functional Kv1.7 channels in mammalian cell
lines that do not express endogenous potassium channels (e.g., CV-1,
NIH-3T3, or RBL cell lines). These cell lines can then be loaded with
.sup.86 Rb (Rb ions permeate through potassium channels nearly as well as
potassium ions) in the presence of absence of extrinsic materials, and
Kv1.7 modifiers identified by their ability to alter .sup.86 Rb-efflux.
When natural toxins are identified which block Kv1.7 activity, modifiers
of Kv1.7 activity could also be identified by their ability to block or
reverse the binding of labeled toxins to cells expressing this channel.
Compounds discovered in either of these manners could then be formulated
and administered as therapeutic agents for the treatment of NIDDM.
C. Materials and Methods
1. Screening of the Mouse Genomic DNA Library
To isolate the Kv1.7 cDNA, approximately 5.times.10.sup.5 plaques from an
AJR/J mouse genomic library were screened (genomic DNA partially digested
with the restriction endonuclease Mbo I and cloned into the vector J1, a
derivative of L47.1) (a gift of Jonathan Kaye, University of California,
San Diego, La Jolla, Calif.). The genomic library was screened using a
mixture of the mouse Kvl.1 (MK1) (Temple et al., Nature 332:837 (1988))
and rat Kvl.5 (KV1) cDNA (Swanson et al., Nature 332:837 (1990)) as a
probe. Probes were labeled with .sup.32 P to a specific activity of
1.times.10.sup.9 cpm/ug by the random primer method of Feinberg and
Vogelstein, Anal. Biochem. 132:6 (1983). The mouse Kvl.1 (MK1) cDNA probe
containing the entire 1485 base pair coding region was obtained from Bruce
Tempel (University of Washington, Seattle, Wash.). The 1.1 kilobase
fragment probe derived from the rat Kvl.5 (KV1) cDNA, containing the
coding region from S3 to its end, was obtained from Leonard Kaczmarek
(Yale University, New Haven, Conn.). Hybridization was performed at
55.degree. C. in hybridization buffer for 16-18 hr. Hybridization buffer
consists of 5.times.SSC, 10.times. Denhardt's (0.2% bovine serum albumin,
0.2% polyvinyl pyrrolidone), and 0.1% SDS. The blots were washed at a
final stringency of 0.5.times.SSC and 0.1% SDS for 60 min at 55.degree. C.
The blots were then exposed to X-OMAT AR film (Kodak, Rochester, N.Y.) at
-70 .degree. C. using an intensifying screen.
DNA was isolated from positive phage clones, digested to completion with
HindIII and electrophoresed on a 0.9% agarose gel. DNA was transferred to
nitrocellulose membranes by capillary transfer and Southern blotting was
performed by the method of Southern, Methods in Enzymology (R. Wu, Ed.),
68:152, Academic Press, New York. Hybridizing and non-hybridizing
fragments were then subcloned into the HindIII site of the pUC19 plasmid
vector.
2. Restriction Mapping
To generate a restriction map of the DNA inserts, plasmid DNA was digested
with from 1-3 restriction enzymes and the order of restriction fragments
assembled from the results. The insert DNA was then sequenced by the
dideoxynucleotide termination method (Sanger et al., Proc. Natl. Acad.
Sci. USA 74:5463 (1977)) and the resultant genomic sequence was aligned
with that of the Shaker-related mouse Kvl.1 (MK1) cDNA. For Southern
blotting experiments, digested DNA fragments were separated by
electrophoresis on a 0.9% agarose gel and then electrotransfered to Nylon
membrane (Nytran, Schleicher & Schuell, Keen, N.H.) using 1.times.
Tris-acetate/EDTA transfer buffer. Electrotransfer was carried out at
4.degree. C. for 14 hrs at 100 mA. Hybridization and washing were carried
out using the same reagents and conditions described above for the library
screening. Exposure of the blots was done on X-OMAT film (Kodak,
Rochester, N.Y.) at room temperature for 30 minutes.
3. DNA Sequencing
A fragment containing a majority of the coding region was cloned into
pBluescript (Stratagene, La Jolla, Calif.), and the inserts were sequenced
by the dideoxynucleotide chain termination method (Sanger et al., Proc.
Natl. Acad. Sci. USA 74:5463 (1977)) using modified T7 DNA polymerase
(Sequenase; US Biochemicals, Cleveland, Ohio). Plasmid-specific primers
and custom designed oligonucleotide primers (purchased from Chemgenes,
Needham, Mass.) were used for this purpose.
4. Northern Blots
For Northern blot analysis, total RNA was isolated by the guanidine
thiocyanate method (Chirgwin et al., Biochemistry 18:5294 (1979)) using
the RNAgents.TM. total RNA isolation kit (Promega, Madison, Wis.). Ten
nanograms of total RNA was fractionated on a 1% agarose gel after
denaturation with glyoxal and dimethyl sulfoxide (McMaster and Carmichael,
Proc. Natl. Acad. Sci. USA 74:4835 (1977)) and was transferred by the
capillary method to nylon membrane (Vrati et al., Mol. Biol. Rep.(Bio-Rad
Laboratories) 1(3):1 (1987)).
A PstI/SacI fragment from the Kv1.7-specific 3' untranslated region of the
cDNA clone was radioactively labeled by the random primer method to a
specific activity of 1.times.10.sup.9 cpm/microgram and used as a probe.
Hybridization was performed at 55.degree. C. in hybridization buffer
consisting of 5.times.SSC, 10' Denhardt's and 0.1% SDS. The blot was then
washed at a final stringency of 0.5.times.SSC and 0.1% SDS for 30 minutes
at 55 .degree. C. and then exposed to X-OMAT film for 72 h at -70 .degree.
C. with an intensifying screen.
5. Polymerase Chain Reaction
Total RNA isolated from mouse brain and from the hamster insulinoma cell
line, HIT-TI5, was used to generate random primed cDNA by the method of
Krug and Berger, Methods in Enzymology (S. L. Berger and A. R. Kimmel,
Eds.) 152:316 (1987) Academic Press, San Diego. The 40 microliter reaction
mixture contained 40 units of avian myeloblastosis virus reverse
transcriptase (Promega, Madison, Wis.), 20 units of RNasin (Promega,
Madison, Wis.), 100 pM random hexanucleotide triphosphate (GeneAmp kit;
Perkin-Elmer-Cetus, Norwalk, Conn.). The cDNA product was then amplified
for 25 cycles with an annealing temperature of 57.degree. C. with TaqI
polymerase (Promega, Madison, Wisconsin) using two oligonucleotide primers
derived from the sequence of the mouse Kv1.7 genomic clone. The upstream
primer 5'-TCTCCGTACTCGTCATCCTGG-3' (SEQ ID NO:20) corresponds to sequence
in the S1 transmembrane segment and the downstream primer
5'-AAATGGGTGTCCACCCGGTC-3'(SEQ ID NO:21) corresponds to the 3'->5'
complementary sequence of the carboxy terminus of the S3--S4 loop of mouse
Kv1.7. The reaction mixture contained 60 mM Tris-HCl pH 8.5, 25 mM
(NH.sub.4).sub.2 SO.sub.4, 2.5 mM MgCl.sub.2, 10% dimethyl sulfoxide, 0.25
microgram of each primer, 2.5 mM of each deoxynucleotide triphosphate and
5 units of TaqI polymerase (Mullis et al., Cold Spring Harbor Symp. Quant.
Biol. 51:263 (1986)).
6. Human Chromosome Localization
Mouse genomic Kv1.7 DNA was used to isolate a human Kv1.7 cosmid clone from
a human chromosome 19-enriched library (Library F) (de Jong et al.,
Cytogen. Cell Genet. 51:985 (1989)), containing an approximately 4.times.
coverage of chromosome 19 as described by Tynan et al., Nucl. Acids Res.
20:1629 (1992) and Tynan et al., Genomics 17:316 (1993). The probe insert
fragment was isolated and labeled by random priming (Feinberg and
Vogelstein, Anal. Biochem. 132:6 (1983)) with .sup.32 P-dCTP for probing.
Fluorescence in situ hybridization (FISH) of cosmids to metaphase
chromosomes was performed as previously described by Trask, Methods Cell
Biol. 35:3 (1990) and Trask et al., Genomics 15:133 (1993). Two color
hybridization to metaphase chromosomes was performed as described by
Brandriff et al., Genomics 12:773 (1992) .
7. Expression Construct
A mouse Kv1.7 expression construct was generated by combining genomic
sequences with PCR-derived cDNA sequences in the pBluescript vector, and
cRNA was prepared and injected into Xenopus oocytes as described by Aiyar
et al., 1993, Amer. J. Physiol. 265:C1571.
8. Materials Testing
The Kv1.7 expression construct described above or related ones expressing
the Kv1.7 potassium channel gene can be used to generate functional
potassium channels in mammalian cell lines that do not express endogenous
potassium channels by transfection of the construct into the cell line.
These cell lines are then loaded with .sup.86 Rb ions which permeate
through potassium channels nearly as well as potassium ions. The loaded
cells can then be cultured in the presence or absence of extrinsic
materials and Kv1.7 channel blockers are identified by their ability to
prevent .sup.86 Rb-efflux. The methods for the above experiments are all
well known in the art.
9. Preparation of antibodies against the Kv1.7 potassium channels
The gene encoding the Kv1.7 potassium channel are isolated by standard
recombinant DNA techniques such as described in Weir et al., Handbook of
Experimental Immunology, Vol. 3 (1986) and other available documents.
These genes are used as templates to prepare Kv1.7 potassium channel
proteins or peptides, which are used as antigens to prepare antibodies
against the Kv1.7 potassium channel. A second method for preparing
antibodies against the Kv1.7 potassium channel protein is used with cells
expressing large numbers of the Kv1.7 channel, isolating the cell surface
proteins from these cells and using these proteins as antigens for the
preparation of antibodies. The antibodies are then screened for the
ability to effect Kv1.7 potassium channels electrophysiologically.
10. Drug and/or antibody testing in Type II diabetes mellitus
Materials comprising drugs or antibodies identified by assays designed to
identify extrinsic materials possessing the ability to modulate the Kv1.7
potassium channel may be tested in vivo for efficacy in appropriate animal
models, for example, for their ability to treat NIDDM by increasing
secretion of insulin from pancreatic .beta.-cells. The route of
administration of the drugs/antibodies can be oral, parental, or via the
rectum, and the drug could be administered alone as principals, or in
combination with other drugs or antibodies, and at regular intervals or as
a single bolus, or as a continuous infusion in standard formations. Drugs
or antibodies described supra are also tested in in vitro assays, for
example, for their ability to stimulate secretion of insulin from
pancreatic .beta.-cells derived from patients or animal models of NIDDM.
11. A treatment protocol
Candidate materials identified by the assays described above are tested for
safety in humans as per Federal guidelines. These candidates described
supra are administered via standard formulations to diseased patients,
again either orally, parenterally, rectally, alone or in combination, at
regular intervals or as a single bolus, or as a continuous infusion, for
modulating Kv1.7 potassium channels in pancreatic .beta.-cells, thereby
impacting on the course of the disease.
The foregoing description details specific methods that can be employed to
practice the present invention. Having detailed specific methods initially
used to identify extrinsic materials possessing the ability to modulate
the Kv1.7 potassium channels on pancreatic .beta.-cells, one skilled in
the art will well enough know how to devise alternative reliable methods
for arriving at the same basic information and for extending this
information to other species including humans. Thus, however detailed the
foregoing may appear in text, it should not be construed as limiting the
overall scope hereof; rather, the ambit of the present invention is to be
governed only by the lawful construction of the appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 21
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCTGCTACTGGCTCGGTTCTTTGTGGTGGAGA32
AlaAlaThrGlySer
15
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AlaAlaThrGlySer
15
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTCCCTTCTGCAGTTCCTCGCCCGA25
PheLeuAlaArg
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
PheLeuAlaArg
1
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCTGCTACTGGCTCGTTCCTCGCCCGA27
AlaAlaThrGlySerPheLeuAlaArg
15
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AlaAlaThrGlySerPheLeuAlaArg
15
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GCTGCTACTGGCTCGTTCCTCTCTCGG27
AlaAlaThrGlySerPheLeuSerArg
15
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AlaAlaThrGlySerPheLeuSerArg
15
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1599 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1599
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGACTACAAGGGAAAGCTCAAGAGATCCACGGAAAAGCGCCGGGTGG48
MetThrThrArgGluSerSerArgAspProArgLysSerAlaGlyTrp
151015
CAGTGTTTCCACAGGTGTGGAACGGCAGAGGGCGCCCCTAGCCCCGCG96
GlnCysPheHisArgCysGlyThrAlaGluGlyAlaProSerProAla
202530
GGGGTAACACCGCCCCCTCCCCCGCGCCCTGGCCGGACTTTCCATGCT144
GlyValThrProProProProProArgProGlyArgThrPheHisAla
354045
ATTTTTACCCGCCGACACCGGACACCCGACTGGGGTGGCTGCGGCGTC192
IlePheThrArgArgHisArgThrProAspTrpGlyGlyCysGlyVal
505560
GGGGCCACACGTCCGTTCACCGGTCGCCCGGGCTGTGCGCGCCATGGA240
GlyAlaThrArgProPheThrGlyArgProGlyCysAlaArgHisGly
65707580
GCCACGGTGCCCGCCGCCCTGCGCTGCTGCGAGCGGCTGGTGCTCAAC288
AlaThrValProAlaAlaLeuArgCysCysGluArgLeuValLeuAsn
859095
GTGGCCGGGTTGCGCTTCGAGACCCGCGCGCGCACGCTCGGCCGCTTC336
ValAlaGlyLeuArgPheGluThrArgAlaArgThrLeuGlyArgPhe
100105110
CCGGACACGCTGCTGGGGGACCCGGTGCGCCGCAGCCGCTTCTACGAC384
ProAspThrLeuLeuGlyAspProValArgArgSerArgPheTyrAsp
115120125
GGCGCGCGCGCCGAGTATTTCTTCGACCGACACCGGCCCAGCTTCGAT432
GlyAlaArgAlaGluTyrPhePheAspArgHisArgProSerPheAsp
130135140
GCGGTGCTCTACTACTACCAGTCGGGCGGCCGGCTGAGACGGCCGGCG480
AlaValLeuTyrTyrTyrGlnSerGlyGlyArgLeuArgArgProAla
145150155160
CACGTGCCCCTCGACGTCTTCCTGGAGGAGGTGTCCTTCTACGGGCTG528
HisValProLeuAspValPheLeuGluGluValSerPheTyrGlyLeu
165170175
GGGCGGCGGCTGGCGCGGCTGCGGGAGGACGAGGGCTGCGCGGTCGCC576
GlyArgArgLeuAlaArgLeuArgGluAspGluGlyCysAlaValAla
180185190
GAGCGGCCGCTGCCCCCGCCCTTTGCGCGTCAGCTCTGGCTGCTCTTC624
GluArgProLeuProProProPheAlaArgGlnLeuTrpLeuLeuPhe
195200205
GAATTTCCTGAGAGCTCGCAGGCTGCGCGCGTGCTCGCCGTGGTCTCC672
GluPheProGluSerSerGlnAlaAlaArgValLeuAlaValValSer
210215220
GTACTCGTCATCCTGGTCTCCATCGTGGTCTTTTGCCTCGAGACACTG720
ValLeuValIleLeuValSerIleValValPheCysLeuGluThrLeu
225230235240
CCAGACTTCCGCGACGACCGCGATGACCCGGGGCTCGCGCCGGTAGCG768
ProAspPheArgAspAspArgAspAspProGlyLeuAlaProValAla
245250255
GCTGCTACTGGCTCGTTCCTCGCTCGGCTCAATGGCTCCAGTCCCATG816
AlaAlaThrGlySerPheLeuAlaArgLeuAsnGlySerSerProMet
260265270
CCAGGAGCCCCTCCCCGACAGCCCTTCAACGATCCATTCTTTGTGGTG864
ProGlyAlaProProArgGlnProPheAsnAspProPhePheValVal
275280285
GAGACCCTGTGTATCTGCTGGTTCTCCTTTGAGCTGCTGGTGCATCTG912
GluThrLeuCysIleCysTrpPheSerPheGluLeuLeuValHisLeu
290295300
GTGGCCTGCCCTAGCAAAGCTGTGTTCTTCAAGAATGTGATGAACCTA960
ValAlaCysProSerLysAlaValPhePheLysAsnValMetAsnLeu
305310315320
ATTGACTTCGTGGCCATCCTGCCTTACTTCGTGGCCCTGGGCACGGAG1008
IleAspPheValAlaIleLeuProTyrPheValAlaLeuGlyThrGlu
325330335
TTAGCCCGGCAGCGGGGTGTGGGCCAGCCGGCTATGTCCCTGGCCATC1056
LeuAlaArgGlnArgGlyValGlyGlnProAlaMetSerLeuAlaIle
340345350
CTAAGGGTCATCCGATTGGTGCGTGTCTTCCGCATCTTCAAGCTCTCC1104
LeuArgValIleArgLeuValArgValPheArgIlePheLysLeuSer
355360365
AGGCATTCGAAGGGTCTACAGATCTTGGGTCAGACACTGCGGGCTTCC1152
ArgHisSerLysGlyLeuGlnIleLeuGlyGlnThrLeuArgAlaSer
370375380
ATGCGTGAGCTAGGTCTCCTCATCTCCTTCCTCTTCATTGGCGTGGTC1200
MetArgGluLeuGlyLeuLeuIleSerPheLeuPheIleGlyValVal
385390395400
CTCTTTTCCAGCGCAGTCTACTTTGCTGAAGTGGACCGGGTGGACACC1248
LeuPheSerSerAlaValTyrPheAlaGluValAspArgValAspThr
405410415
CATTTCACCAGCATCCCGGAGTCCTTTTGGTGGGCAGTGGTCACCATG1296
HisPheThrSerIleProGluSerPheTrpTrpAlaValValThrMet
420425430
ACCACGGTTGGCTATGGGGACATGGCACCCGTCACCGTGGGTGGCAAG1344
ThrThrValGlyTyrGlyAspMetAlaProValThrValGlyGlyLys
435440445
ATCGTGGGCTCTCTGTGTGCCATTGCAGGTGTGCTCACCATCTCTCTG1392
IleValGlySerLeuCysAlaIleAlaGlyValLeuThrIleSerLeu
450455460
CCTGTGCCTGTCATTGTCTCTAACTTTAGCTACTTTTACCACCGGGAG1440
ProValProValIleValSerAsnPheSerTyrPheTyrHisArgGlu
465470475480
ACAGAGGGCGAAGAGGCAGGGATGTACAGCCATGTGGACACACAGCCC1488
ThrGluGlyGluGluAlaGlyMetTyrSerHisValAspThrGlnPro
485490495
TGCGGTACCCTGGAGGGCAAGGCTAATGGGGGGCTGGTGGACTCTGAG1536
CysGlyThrLeuGluGlyLysAlaAsnGlyGlyLeuValAspSerGlu
500505510
GTGCCTGAACTCCTCCCACCACTCTGGCCCCCTGCAGGGAAACACATG1584
ValProGluLeuLeuProProLeuTrpProProAlaGlyLysHisMet
515520525
GTGACTGAGGTGTGA1599
ValThrGluVal
530
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
MetThrThrArgGluSerSerArgAspProArgLysSerAlaGlyTrp
151015
GlnCysPheHisArgCysGlyThrAlaGluGlyAlaProSerProAla
202530
GlyValThrProProProProProArgProGlyArgThrPheHisAla
354045
IlePheThrArgArgHisArgThrProAspTrpGlyGlyCysGlyVal
505560
GlyAlaThrArgProPheThrGlyArgProGlyCysAlaArgHisGly
65707580
AlaThrValProAlaAlaLeuArgCysCysGluArgLeuValLeuAsn
859095
ValAlaGlyLeuArgPheGluThrArgAlaArgThrLeuGlyArgPhe
100105110
ProAspThrLeuLeuGlyAspProValArgArgSerArgPheTyrAsp
115120125
GlyAlaArgAlaGluTyrPhePheAspArgHisArgProSerPheAsp
130135140
AlaValLeuTyrTyrTyrGlnSerGlyGlyArgLeuArgArgProAla
145150155160
HisValProLeuAspValPheLeuGluGluValSerPheTyrGlyLeu
165170175
GlyArgArgLeuAlaArgLeuArgGluAspGluGlyCysAlaValAla
180185190
GluArgProLeuProProProPheAlaArgGlnLeuTrpLeuLeuPhe
195200205
GluPheProGluSerSerGlnAlaAlaArgValLeuAlaValValSer
210215220
ValLeuValIleLeuValSerIleValValPheCysLeuGluThrLeu
225230235240
ProAspPheArgAspAspArgAspAspProGlyLeuAlaProValAla
245250255
AlaAlaThrGlySerPheLeuAlaArgLeuAsnGlySerSerProMet
260265270
ProGlyAlaProProArgGlnProPheAsnAspProPhePheValVal
275280285
GluThrLeuCysIleCysTrpPheSerPheGluLeuLeuValHisLeu
290295300
ValAlaCysProSerLysAlaValPhePheLysAsnValMetAsnLeu
305310315320
IleAspPheValAlaIleLeuProTyrPheValAlaLeuGlyThrGlu
325330335
LeuAlaArgGlnArgGlyValGlyGlnProAlaMetSerLeuAlaIle
340345350
LeuArgValIleArgLeuValArgValPheArgIlePheLysLeuSer
355360365
ArgHisSerLysGlyLeuGlnIleLeuGlyGlnThrLeuArgAlaSer
370375380
MetArgGluLeuGlyLeuLeuIleSerPheLeuPheIleGlyValVal
385390395400
LeuPheSerSerAlaValTyrPheAlaGluValAspArgValAspThr
405410415
HisPheThrSerIleProGluSerPheTrpTrpAlaValValThrMet
420425430
ThrThrValGlyTyrGlyAspMetAlaProValThrValGlyGlyLys
435440445
IleValGlySerLeuCysAlaIleAlaGlyValLeuThrIleSerLeu
450455460
ProValProValIleValSerAsnPheSerTyrPheTyrHisArgGlu
465470475480
ThrGluGlyGluGluAlaGlyMetTyrSerHisValAspThrGlnPro
485490495
CysGlyThrLeuGluGlyLysAlaAsnGlyGlyLeuValAspSerGlu
500505510
ValProGluLeuLeuProProLeuTrpProProAlaGlyLysHisMet
515520525
ValThrGluVal
530
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTATTTTTACGNGCGGACACCGGACTACCG30
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGCTGGGGCGGCGGNGG17
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TGCTCGTCCGTAGTCTCCGTGCTCCTCATCCTCGTCTCCATCGTCGTCTTCTGCCTCGAG60
ACGCTGCCT69
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCCGACTCCGCTGAATGGCTCCCAGCC27
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
ATTCTTTGTGGTGGAACCTTTGT23
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ATCTGCTGGTTCTCCTTTGAGCATGCTGGTGCGTCTGGCGGCGTGTCCAAGCAAAGCTGT60
ATTTTTCAAGAATGTGATGAACCTTATTGACTT93
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GTGGCCATCCTGCCTTACTTTGTGGCCCTGGGCACAGAGTTAGCC45
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GTCAGCGGGGCGTGGGCCAGCCAGCTATGTCCCTGGCCATCCTGAGGAGTCATCNGATTG60
GTGCGTAGTCTTCCGCATCTTCAAGCTNTCCNGGCANTCNAAGGGCNTGCAAATCTTGGG120
CCAGGACGCTTCGGGCCTCCATGCGTGAAGCTGGGCCTCCTCATCTTTTTCCTCTTCATC180
GGTGTGGTCCTCTTTT196
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
TTTCCCTGCCAGTGCCCGTCATTGTCTCCAATTTCAGCTACTTTTATCACCGGGAGACAG60
AGGGCGAAGAGGCTGGGATGTTCAGCCATGTGGACATGCAGCCTTGTGGCCCACTGGANG120
GNNCANGNCNANNCCAATGGGGGGCTGGTGGACGGGGAGGTACCTGAGCTACCACCTCCA180
CTCTGGGCACCCCCAGGGAAACACCTGGTCACCGAAGTGTGAGGAACAGTTGAGGTCTGC240
AGGAATTCGATATCAAGCTTATCGATACCGT271
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
TCTCCGTACTCGTCATCCTGG21
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
AAATGGGTGTCCACCCGGTC20
__________________________________________________________________________
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